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Determination of prothrombin activation fragments in young patients with inflammatory bowel disease.

OBJECTIVE: To assess coagulation cascade activation as a potential index of thromboembolic risk in inflammatory bowel disease (IBD). STUDY DESIGN: Fifty plasma samples were obtained during consecutive outpatient encounters with 29 patients (male:female = 20:9) with either Crohn's disease (CD) (n = 23) or ulcerative colitis (UC) (n = 6). Disease activity for CD was determined using the Pediatric Crohn's Disease Activity Index (PCDAI) and for UC using signs/symptoms and mucosal histology. Patients were grouped as active, recently active (not currently active but had been active within the previous 2 months), or inactive. Prothrombin fragment 1.2 (F1.2) was determined by enzyme-linked immunosorbent assay on plasma samples. Lab parameters were compared using the Kruskal-Wallis test with pairwise comparisons made using Wilcoxian rank sum test. RESULTS: Forty-three percent of samples taken during active phases of disease (13 of 30) had elevated prothrombin cleavage byproduct F1.2. Similarly, five of eight encounters (63%) with recently active IBD had elevated F1.2 values. In contrast, none of the patients with inactive disease exhibited F1.2 elevation. Median F1.2 levels in both the active (0.85 nM/L) and recently active (1.4 nM/L) patient groups were greater than that of the inactive group (0.6 nM/L; p < 0.05). CONCLUSIONS: Evidence of coagulation cascade activation in patients with active and recently active IBD suggests an increased risk of thrombogenesis during active disease that extends for a period of time after commencement of medications to induce remission in their disease process. It may be prudent to counsel patients to avoid risk factors associated with thrombotic phenomenon around the time of active IBD.

Adolescent↗

Down-regulation of protein kinase C isoforms in irritant contact dermatitis.

Protein kinase C (PKC) isoforms are important in cell signal transduction associated with regulation of cell proliferation and differentiation. In this study, alterations of PKC isoform levels in irritant patch test reactions were detected by Western immunoblots. 4 chemically and structurally different irritants, 4% and 8% hydrochloric acid (HCl); 1% and 2% sodium hydroxide (NaOH); 20% and 40% nonanoic acid (NON) in 1-propanol; and 5% and 10% sodium dodecyl sulfate (SDS) were applied on the backs of mice in Finn Chambers and fixed with surgical dressings. The patches were kept on the skin for 24 h and removed. 24 h after removal, mild erythema with thickening of skin without vesicles was observed in all irritated skin, except that 4% HCl and 1% NaOH treated skin showed unremarkable skin reaction. No visible skin reaction was detected in vehicle-treated skin. PKC isoform alpha, beta, gamma, and delta levels in irritated skin revealed a 10% to 65% decrease compared to vehicle treated skin. These results indicate that in HCl, NaOH, NON and SDS-induced irritation, activation of the PKC related cell signal transduction cascade may be involved, and that PKC mediated events may be a common phenomenon in irritant contact dermatitis.

Animals↗

The -822G/A polymorphism in the promoter region of the MAP4K5 gene is associated with reduced risk of type 2 diabetes in Chinese Hans from Shanghai.

MAP4K5 (mitogen-activated protein kinase kinase kinase kinase 5), an early component of MAP kinase signal cascades was shown to activate Jun kinase in mammalian cells. The association between SNPs of MAP4K5 and type 2 diabetes (T2DM) was investigated due to the known relationship of the JNK pathway with T2DM. A total of 1,399 cases were included in the study. Oral glucose tolerance test (OGTT) and insulin release test (IRT) were performed, and blood DNA samples were extracted and genotyped on the MAP4K5 -822G/A site. These cases were subdivided into central-obesity and nonobesity groups, based upon their individual waist circumference. Allele-specific real-time PCR was employed for genotyping. No difference was found between the two groups in the distribution of three genotypes on MAP4K5 -822G/A. In the central-obesity group, fewer diabetic patients (38.9%) were present in the AA genotype group than the GG/GA group (58.5%, P=0.024). Glucose levels after 30 and 60 min of 75 g glucose tolerance, area under the curve for glucose, and insulin secretion indexes were lower (P<0.05) in AA than those in GG/GA genotype group in the central-obesity cases. Other variables did not show significant differences between the two groups. In the Han population from Shanghai, the AA genotype of MAP4K5 -822G/A in central-obesity cases appears less likely to develop diabetes compared with the other genotypes. Therefore, the G allele may be a factor that does not protect central-obesity cases from developing into diabetes.

Adult↗

Effects of serum versus plasma on agglutination of antibody-coated indicator cells by human rheumatoid factors.

Plasma and serum contain an inhibitor (I) of the agglutination of rabbit IgG-coated sheep erythrocytes by human rheumatoid factor (RF). Plasma but not serum contains an inhibitor of the inhibitor (I/I) which allows RF to interact with its target. In normal blood, there is more I than I/I, and I can be removed by solid-phase chromatography through concanavalin A (Con A). Plasma I/I is heat labile being eliminated by heating at 56 degrees C for 30 min. Addition of exogenous calcium clots EDTA plasma, causing an irreversible loss of I/I, and suggesting its involvement in the clotting cascade. The absence of I/I from Factor V-deficient plasma and destruction of I/I by Russell's viper venom indicate I/I either is associated with or is a part of Factor V. These findings suggest a balanced interplay between I and I/I, and indicate results of immunological tests done in vitro may not accurately reflect immune function in vivo. This seems to represent an unexplored link between hemostasis and immunity.

Agglutination Tests↗

Recovery of prostacyclin capacity of irradiated endothelial cells and the protective effect of vitamin C.

Ionizing irradiation has been reported to affect prostacyclin (PGI2) production by intact blood vessels and cultured endothelial cells (EC) due to damage of enzymes of the arachidonate cascade. In the present study, we investigated whether EC can recover from radiation injury and regain their capacity to produce PGI2. Bovine aortic EC were exposed to radiation doses of 3 and 6 Gy and their capacity to produce PGI2 in response to stimulation with arachidonic acid was tested, at various times after irradiation. The results of these experiments showed clearly that EC exposed to single or fractionated irradiation could recover their capacity to produce PGI2 depending on the radiation dose and the time period following radiation. Radiation damage is associated with oxidant stress and the production of free radicals. We therefore tested the ability of an oxygen radical scavenger, vitamin C, to protect the capacity of irradiated EC to produce PGI2. Pretreatment of EC with low concentrations of vitamin C inhibited the radiation induced release of PGI2 to the culture medium. Vitamin C also enhanced the capacity of irradiated EC to produce PGI2 following short stimulation with arachidonic acid. Treatment with this scavenger however, did not protect the cells against the cytopathic effects of radiation.

Animals↗

The cytokine receptor superfamily.

The binding of haemopoietic growth factors and cytokines to specific receptors triggers a cascade of intracellular events which results in cell proliferation and differentiation. The knowledge of ligand-receptor-signal pathways is not only important in understanding the pathophysiology of malignant disease but also essential for devising future therapeutic strategies. The advent of recombinant technology has made it possible to test the efficacy of selective differentiation therapy, and haemopoietic growth factors are undergoing clinical trials for a number of indications. In addition, increasingly the receptors for haemopoietic growth factors and cytokines have come under scientific scrutiny. Recently receptors for IL-2 alpha, IL-2 beta, IL-3, IL-4, IL-5, IL-6, IL-7, erythropoietin, G-CSF and GM-CSF have been isolated and cloned. It has become apparent that they have structural homology that is shared by receptors for growth hormone and prolactin, and this receptor group makes up the new cytokine receptor superfamily. The finding of sequence homology within these receptors suggests their evolutionary relationship. These receptors are transmembrane proteins 257-856 amino acids and their extracellular ligand-binding domain contains four conserved cysteine residues and a Trp-Ser-X-Trp-Ser motif. The secondary structure of the extracellular domain is made up of alpha-helices. High and low affinity binding forms exist for all these receptors. Binding affinity may depend on the formation of receptor heterodimers or multimers, association with other membrane proteins or differential glycosylation. Soluble receptor forms have been described for IL-2 alpha, IL-4, IL-5, IL-6 and IL-7. It is not known whether they are actively secreted or represent the degradation products of cell turnover. Their function may be to mop up excess cytokines and thereby confine the cytokine response. There is no sequence homology of the intracytoplasmic domains although several are rich in proline and serine residues, which may be important in mechanisms of signal transduction. No receptor in this superfamily functions as a receptor tyrosine kinase or has intrinsic protein tyrosine kinase activity. Detailed study of individual receptors holds clues to the regulation of receptor expression, ligand-receptor interactions and mechanisms involved in signal transduction. Such knowledge might explain the pleotropic effects cytokines may have on different cell types and their overlap in biological functions. Elevated levels of soluble IL-2 alpha receptor (Tac) are detected in hairy cell leukaemia, lymphomas and adult T-cell leukaemia (TL), and levels reflect tumour burden.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

Hedgehog signaling plays a conserved role in inhibiting fat formation.

Hedgehog (Hh) signals regulate invertebrate and vertebrate development, yet the role of the cascade in adipose development was undefined. To analyze a potential function, we turned to Drosophila and mammalian models. Fat-body-specific transgenic activation of Hh signaling inhibits fly fat formation. Conversely, fat-body-specific Hh blockade stimulated fly fat formation. In mammalian models, sufficiency and necessity tests showed that Hh signaling also inhibits mammalian adipogenesis. Hh signals elicit this function early in adipogenesis, upstream of PPARgamma, potentially diverting preadipocytes as well as multipotent mesenchymal prescursors away from adipogenesis and toward osteogenesis. Hh may elicit these effects by inducing the expression of antiadipogenic transcription factors such as Gata2. These data support the notion that Hh signaling plays a conserved role, from invertebrates to vertebrates, in inhibiting fat formation and highlighting the potential of the Hh pathway as a therapeutic target for osteoporosis, lipodystrophy, diabetes, and obesity.

3T3-L1 Cells↗

Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella.

Two hemolymph proteins were isolated from the wax moth, Galleria mellonella, larvae by a two-step procedure consisting of acid extraction and reversed phase (RP)-HPLC. One was an apolipophorin III (apoLp-III) previously characterized as a lipopolysaccharide (LPS) binding protein in the hemolymph of G. mellonella. The other was confirmed to be a new protein with a molecular mass of 23,768.69 Da, referred to as Gm protein-24. The full-length cDNA of Gm protein-24 was cloned from the fat body. The cDNA structure showed that it is a 219-residues protein derived from the precursor of 236 amino acids. The effects of apoLp-III and Gm protein-24 have been tested on the insect humoral immunity. ApoLp-III enhanced the activity of antibacterial peptide such as cecropin but Gm protein-24 had no effect on cecropin activity. On the other hand, Gm protein-24 and apoLp-III were both involved in the activation of prophenoloxidase (PPO) cascade, which has been regarded as a critical immune reaction in insect hemolymph. Of note, the Gm protein-24 was a significantly stronger activator of PPO cascade than apoLp-III.

Amino Acid Sequence↗

Increased expression levels of integrin alphavbeta5 on scleroderma fibroblasts.

Integrin alphavbeta5 is a receptor for vitronectin, a plasma glycoprotein that is also distributed in extracellular matrix of various tissues. Matrix-bound vitronectin has the potential to stabilize the active form of plasminogen activator inhibitor-1, resulting in the inhibition of the plasmin-mediated pericellular proteolytic cascade. In this study, we compared the levels of alphavbeta5 and matrix-bound vitronectin between normal and scleroderma fibroblasts and investigated the association with fibrosis. We demonstrated that alphavbeta5 was up-regulated on scleroderma fibroblasts. The up-regulated alphavbeta5 contributed to the increase in vitronectin-binding ability in scleroderma fibroblasts, which led to the vitronectin-dependent activation of plasminogen activator inhibitor-1. In immunohistochemistry, the alphav and beta5 subunits were stained strongly on scleroderma fibroblasts and the amount of vitronectin was increased in the pericellular matrix of those cells. The transient overexpression of alphavbeta5 on normal fibroblasts enhanced the human alpha2(I) collagen promoter activity through Sp-1 and Smad3 as well as the vitronectin-dependent plasminogen activator inhibitor-1 activity. This effect on the promoter activity was also observed in the absence of vitronectin and completely disappeared in the presence of anti-alphavbeta5 antibody. These results indicate that the up-regulated alphavbeta5 may contribute to the phenotypical alteration of scleroderma fibroblasts, while at the same time suppressing the plasmin-mediated pericellular proteolytic cascade.

Blotting, Northern↗

Effect of hypoxia and endothelial loss on vascular smooth muscle cell responsiveness to VEGF-A: role of flt-1/VEGF-receptor-1.

OBJECTIVE: The influence of hypoxia and endothelial loss on the responsiveness of vascular smooth muscle cells (VSMCs) to vascular endothelial growth factor (VEGF-A) was tested. METHODS AND RESULTS: Exposure to hypoxia induced a potentiation of cultured cell proliferation in response to either the agonist for the VEGF receptor 1 (flt-1) placental growth factor (PlGF-1) or to VEGF-A. This effect was mediated by the mitogen activated protein kinase (MAPK) cascade, since it was inhibited by the MAPK kinase inhibitor PD98059 and by the farnesyl transferase inhibitor II. Accordingly, PlGF-1 activated extracellular signal-regulated kinase(1/2). In rat aortic rings deprived of endothelium and cultured in three-dimensional fibrin gels, an increased sprouting of tubular structures in response to VEGF-A was observed only under hypoxia. Studies on rat aorta preparations revealed an endothelium-dependent vasorelaxation in response to either VEGF-A or PlGF1, which was reversed to a contractile response in endothelium-deprived preparations exposed to hypoxia. Western blot and immunohistochemistry of endothelium-deprived preparations exposed to hypoxia showed flt-1 receptor expression in all medial cells. Conversely, flt-1 mRNA, of endothelium-deprived aortic preparations and of tubular structures, was unchanged by hypoxia. CONCLUSION: These findings demonstrate that experimental conditions mimicking pathological vascular injury can make VSMCs responsive to VEGF-A through the induction of functional flt-1 receptors.

Animals↗

The effect of phorbol esters on spinal cord amino acid concentrations and responsiveness of rats to mechanical and thermal stimuli.

There is now mounting evidence supporting the hypothesis that pathological perceptual disorders described as secondary hyperalgesia and allodynia may be due to sensitization of spinal cord dorsal horn neurons. Protein kinase C (PKC) is thought to be one of the factors in the cascade of events leading from peripheral tissue damage to the sensitization of central neurons. In our experiments, we have used local microdialysis administration of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to activate PKC in the spinal cord dorsal horn in awake rats. In behavioral tests the responsiveness of the animals to von Frey filaments (1-1200 mN) and to heat stimuli applied to the hindpaws was tested. Thirty minutes after the TPA infusion the threshold for the paw withdrawal response was significantly decreased (from 160 to 6 mN) and the responses to suprathreshold stimuli were more robust. An increased mechanical sensitivity was no longer present when tested 1.5 and 5 h after the TPA application was terminated. When heat stimuli were tested, the TPA infusion resulted in a significantly prolonged time during which the animals held their hindpaws above the supporting surface after the heat stimulus (0.5 and 1.5 h after TPA), and in a decreased threshold for the heat stimulus (latency of withdrawal) 5 h after TPA. HPLC analysis of the perfusate obtained by microdialysis in vivo showed a significant increase in the extracellular levels of aspartate, glutamate, glycine and taurine, and a decrease of the glutamine level during TPA infusion. The levels of asparagine, serine, threonine and alanine did not change. Application of the inactive phorbol ester (alpha-TPA) did not evoke any change from the control values either in the AA concentrations or in the behavioral tests. Our results suggest that activation of PKC in the spinal cord evokes mechanical allodynia and thermal hyperalgesia and provides further evidence that PKC is involved in the process of the modulation of nociceptive information at the spinal cord level.

Amino Acids↗

Transformation of Leishmania mexicana metacyclic promastigotes to amastigote-like forms mediated by binding of human C-reactive protein.

Infective metacyclic promastigote forms of Leishmania mexicana are introduced by the bite of sandfly vectors into their human hosts where they transform into the amastigote form. The kinetics of this process was examined in vitro in response to different combinations of temperature (26 degrees C or 32 degrees C), pH (7.2 or 5.5), and exposure to human serum. Little transformation occurred at 26 degrees C/pH 7.2, intermediate levels at 26 degrees C/pH 5.5 and 32 degrees C/pH 7.2, and the greatest response at 32 degrees C/pH 5.5. Transformation was stimulated by exposure to normal human serum, but was markedly reduced when serum previously incubated at 56 degrees C for 1 h was used (complement heat-inactivated). This stimulatory effect was reproduced by exposure to a single purified component of human serum, C-reactive protein (CRP). Binding of CRP to the whole surface of L. mexicana metacyclic promastigotes, including the flagella, was demonstrated by an indirect fluorescent antibody test. The effect of purified CRP was dose dependent and occurred using normal serum concentrations. The stimulatory effect of whole serum was oblated by CRP depletion and restored by addition of purified CRP. The effects of cAMP analogues indicated that transformation could be mediated via an adenylate cyclase cascade.

Adenylyl Cyclases↗

In search of the digitalis replacement.

There are now several new CT drugs undergoing clinical study for the treatment of CHF. While most of these new agents are like digitalis in that they possess significant inotropic activity, they differ from digitalis in that they tend to also have pronounced vasodilator properties. Such compounds, because they are not pure CT agents, should not be regarded as true digitalis replacements. Despite the increased understanding about the cAMP cascade and about cardiac muscle contraction in general, the long search for a specific digitalis replacement cannot be regarded as having been accomplished. It should also be noted that the initial clinical assessment for any of these new compounds will be complicated by the fact that they are tested in only the latter stages of CHF where prognosis is very poor and the possibility of showing significant improvement in mortality is extremely difficult. It will be unfortunate if the potential for positive inotropic agents (pure CT drugs) is compromised by clinical studies that employ mixed inotropic-vasodilator compounds in extremely sick patient populations. The continued use of the digitalis glycosides for such a long period of history implies that at least some subpopulation of CHF patients should derive benefit from therapy with an appropriately selective CT drug. Neither the appropriate drug nor the appropriate specific patient population, both of which are needed to properly address the eventual role of inotropic therapy in CHF, have, as yet, been identified.

3',5'-Cyclic-AMP Phosphodiesterases↗

Chitin catabolism in the marine bacterium Vibrio furnissii. Identification, molecular cloning, and characterization of A N, N'-diacetylchitobiose phosphorylase.

The major product of bacterial chitinases is N,N'-diacetylchitobiose or (GlcNAc)(2). We have previously demonstrated that (GlcNAc)(2) is taken up unchanged by a specific permease in Vibrio furnissii (unlike Escherichia coli). It is generally held that marine Vibrios further metabolize cytoplasmic (GlcNAc)(2) by hydrolyzing it to two GlcNAcs (i.e. a "chitobiase "). Here we report instead that V. furnissii expresses a novel phosphorylase. The gene, chbP, was cloned into E. coli; the enzyme, ChbP, was purified to apparent homogeneity, and characterized kinetically. The DNA sequence indicates that chbP encodes an 89-kDa protein. The enzymatic reaction was characterized as follows. (GlcNAc)(2)+P(i) GlcNAc-alpha-1-P+GlcNAc K'(cq)=1.0+/-0.2 Reaction 1 The K(m) values for the four substrates were in the range 0.3-1 mm. p-Nitrophenyl-(GlcNAc)(2) was cleaved at 8.5% the rate of (GlcNAc)(2), and p-nitrophenyl (PNP)-GlcNAc was 36% as active as GlcNAc in the reverse direction. All other compounds tested displayed </=1% of the activity of the indicated substrates including: for phosphorolysis, higher chitin oliogsaccharides, (GlcNAc)(n), n = 3-5, cellobiose, PNP-GlcNAc, and PNP-(GlcNAc)(3); for synthesis, (GlcNAc)(n) (n = 2-5), glucose, etc. (GlcNAc)(2) is a major regulator of the chitin catabolic cascade. Conceivably GlcNAc-alpha-1-P plays a similar but different role in regulation.

Acetylglucosamine↗

c-Jun associates with the oncoprotein Ski and suppresses Smad2 transcriptional activity.

The Smad proteins are key intracellular effectors of transforming growth factor-beta (TGF-beta) cytokines. The ability of Smads to modulate transcription results from a functional cooperativity with the coactivators p300/cAMP-response element-binding protein-binding protein (CBP), or the corepressors TGIF and Ski. The c-Jun N-terminal kinase (JNK) pathway, another downstream target activated by TGF-beta receptors, has also been suggested to inhibit TGF-beta signaling through interaction of c-Jun with Smad2 and Smad3. Here we show that c-Jun directly interacts with Ski to enhance the association of Ski with Smad2 in the basal state. Interestingly, TGF-beta signaling induces dissociation of c-Jun from Ski, thereby relieving active repression by c-Jun. Moreover, activation of JNK pathway suppressed the ability of TGF-beta to induce dissociation of c-Jun from ski. Thus, the formation of a c-Jun/Ski complex maintains the repressed state of Smad2-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-beta signaling by the JNK cascade.

Animals↗

In vitro effect of a holding chamber on the mouth-throat deposition of QVAR (hydrofluoroalkane-beclomethasone dipropionate).

Experimental work has been conducted on the effect of an add-on holding chamber (Aerochamber) on the characteristics of deposition in a mouth-throat model using 100-microg hydrofluoroalkane-beclomethazone dipropionate (QVAR) metered dose inhalers at inhalation flow rates of 28.3, 60, and 90 L/min. A filter or cascade impactor downstream of the mouth-throat collected aerosol not depositing. The results emphasize the important well documented effect of a valved holding chamber (VHC), in reducing drug deposition in the mouth-throat. This reduction is largest (24% of nominal dose) at the lowest flow rate tested, becoming insignificant at 60 L/min. Total amount of drug delivered distal to the mouth-throat increases with flow rate both with and without a holding chamber, increasing from 42% to 69% of the nominal dose without a VHC as the inspiratory flow rate increases from 28.3 to 90 L/min. The effect of the holding chamber on post mouth-throat delivery was small, reaching significance only at the highest flow rate (90 L/min), where an increase by 8% of the nominal dose was observed. No significant effect on MMAD of beclomethasone-dipropionate occurred when the holding chamber was used. An argument based on the interaction between induced turbulence and particle inertia is used to shed light on the above observations.

Aerosols↗

Mannose-binding protein recognizes glioma cells: in vitro analysis of complement activation on glioma cells via the lectin pathway.

The lectin pathway is a novel pathway for activation of the complement cascade, which is initiated by the binding of mannose-binding protein (MBP) to its carbohydrate ligands. We investigated whether the complement system was activated in vitro by glioma cells through this pathway to the C3 level. MBP was found to bind to all six glioma cell lines tested by using flow cytometric analysis. Binding of a complex of MBP-associated serine protease and MBP was observed in two of the cell lines examined, thereby resulting in C4 consumption. Activation of C3 was hemolytically evaluated in these two lines. C3 consumption was also observed in one. Based on these results, it is likely that recognition by MBP followed by complement activation occurs in certain glioma cell lines.

Carrier Proteins↗

Incretin effect potentiates beta-cell responsivity to glucose as well as to its rate of change: OGTT and matched intravenous study.

The aim of this study is to gain greater insight into the mechanism whereby "incretins" (greater insulinemia after oral than intravenous glucose) enhance insulin secretion. To do so, we use a model of C-peptide secretion to reanalyze data from a previously published study in which glycemic profiles observed following glucose ingestion were matched in the same 10 subjects by means of an intravenous glucose infusion. We report that incretins increase insulin secretion by enhancing both the dynamic (to the rate of increase of glucose) and static (to given glucose concentration) response with an increase of 58% for the static (Phi(s) = 16.4 +/- 1.8 vs. 24.6 +/- 2.0 10(-9) min(-1), P = 0.01) and 63% for the dynamic (Phi(d) = 278 +/- 32 vs. 463 +/- 86 10(-9), P = 0.02) indexes. Since increases in the dynamic response to glucose are believed to be due to an increase in the rate of docking, and exocytosis of insulin containing granules and increases in the static response to glucose are believed to be caused by a shift in the sensitivity of the beta-cell to glucose, these results suggest that incretins may modulate more than one step in the beta-cell insulin secretory cascade.

Administration, Oral↗